Amyloid plaques are a major pathological hallmark of Alzheimer's disease (AD). The beta-amyloid peptide (Abeta) is the main component of amyloid plaques, and evidence suggests that Abeta is central to AD pathogenesis. Abeta is generated from the amyloid precursor protein (APP) via sequential cleavage by two proteases, the beta- and gamma-secretases. The beta- secretase, a novel aspartic protease termed BACE1, was initially cloned and characterized in our group. BACE1 initiates the formation of Abeta and therefore is a key drug target for the treatment of AD. The recent generation of BACE1-/- mice by gene targeting in our group has validated BACE1 as the authentic beta-secretase in vivo. In AD, amyloid plaques are associated with a focal inflammatory response that involves activated glia. Inflammatory markers are dramatically elevated in the brains of AD patients and amyloid plaque-developing APP transgenic mice. IL-1beta is a major cytokine released during AD, and recent studies demonstrate that IL-1beta increases the synthesis of APP in cultured astrocytes. Moreover, in APP transgenic mice, activated astrocytes around plaques express BACE1. These data suggest that activated astrocytes express both APP substrate and BACE1 enzyme and thus may produce significant amounts of Abeta. Our hypothesis is that activated astrocytes surrounding amyloid plaques exacerbate plaque development by generating Abeta during AD inflammation. This hypothesis is supported by evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of AD, and that the NSAID ibuprofen inhibits amyloid plaque development in APP transgenic mice. The long-term goal of this project is to understand the role of activated astrocytes in Abeta production and amyloid plaque development. The Specific Aims are the following: 1.) Determine whether BACE1 and APP levels are increased in activated astrocytes and, if so, whether activated astrocytes exhibit increased Abeta production and contribute to amyloid deposition; 2.) Determine whether IL-1beta causes increases in APP and BACE1 synthesis and Abeta production in astrocytes and neurons in vitro and in vivo, and facilitates amyloid deposition in vivo. We will use cultured mouse astrocytes and transgenic and knockout mice as our main model systems for this project. Our analytical approaches will include a combination of molecular (Northern, Western, immunoprecipitation, ELISA) and cellular (immunohistochemistry/light microscopy, immunofluoescence/confocal microscopy) techniques. Importantly, we will develop novel APP transgenic models and use the IL-1beta pellet implantation model of Griffin and colleagues in this project. We anticipate that these studies will provide critical insight into the role of astrocytes and BACE1 in Abeta generation and amyloid deposition, mechanisms that are central to AD pathophysiology.